Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics

Ammonia release from proteinaceous feedstocks represents the main inhibitor of the anaerobic digestion (AD) process, which can result in a decreased biomethane yield or even complete failure of the process. The present study focused on the adaptation of mesophilic AD communities to a stepwise increase in the concentration of ammonium chloride in synthetic medium with casein used as the carbon source. An adaptation process occurring over more than 20 months allowed batch reactors to reach up to 20 g of NH4+ N/L without collapsing in acidification nor ceasing methane production. To decipher the microbial dynamics occurring during the adaptation and determine the genes mostly exposed to selective pressure, a combination of biochemical and metagenomics analyses was performed, reconstructing the strains of key species and tracking them over time. Subsequently, the adaptive metabolic mechanisms were delineated by following the single nucleotide variants (SNVs) characterizing the strains and prioritizing the associated genes according to their function. An in-depth exploration of the archaeon Methanoculleus bourgensis vb3066 and the putative syntrophic acetate-oxidizing bacteria Acetomicrobium sp. ma133 identified positively selected SNVs on genes involved in stress adaptation. The intraspecies diversity with multiple coexisting strains in a temporal succession pattern allows us to detect the presence of an additional level of diversity within the microbial community beyond the species level.

Table S1       *Starting from the 10 th generation, two different concentrations have been adopted in parallel

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(distinguished by the presence of an "s" after the number).

Section S6: Glucose-and Acetate-fed reactors performances
In the case of glucose as the main C source, a drop to zero in CH 4 production suggested a system failure.Starting from the 4 th generation, the acidification of the medium occurred, coherently with the absence of methane production.Frequently, a drop in pH is associated with an accumulation of short-chain fatty acids determined by a faster metabolic activity of the hydrolytic over acetoclastic species (the primary degraders), in comparison to the transformation rate accomplished by the acetoclastic species.As a consequence, there is a lack of molecules for methanogenesis, resulting in AD failure.For this reason, glucose batches were halted at the 4 th generation.In the acetate-fed cultures, the pH remained stable, but a system failure occurred in the 6 th and 7 th generations, where no methane production was detected.
Different explanations for the selection of low ammonia-resistant species in glucose and acetate-fed batches can be formulated.First, the drastic change in substrate composition could have led to a major loss of protein-degrading species, while the surviving microbes were not able to cope with the increased ammonia concentration.Second, the delicate acetoclastic archaea were the most likely to be negatively selected aiding the system failure.Interestingly, in casein-fed communities, a high and protracted resilience was observed.Noteworthy, methanation occurred still in the last reached generation (27 th ), although with a very limited cumulative production of methane observed.

Section S7: Metagenomic and variants results
Long and short reads were combined to obtain a high-quality hybrid assembly, for a total length of 618.24 Mb.The average alignment rate of short reads on the assembly was 96.78%, confirming that the microbial community is almost entirely represented.After the binning, 70.69% of the reads were aligned to the MAGs, with values ranging from 56.84 to 80.44% according to the sample.This indicates that the following taxonomic and functional analyses performed on MAGs accurately represent the majority of the microbiome.The integration of multiple binning approaches allowed the identification of 179 MAGs, 99 of them of high quality and 80 of medium quality, according to the minimum information about metagenomeassembled genomes (MIMAG) 26 .A global analysis previously performed on AD systems revealed an average assembly size of 409 Mbp (minimum 47 Mbp, maximum 2.3 Gbp) and an average number of MAGs of 148 (minimum 20, maximum 636 MAGs) for the AD microbiome 27 .This suggests that the community of the current study has an intermediate level of complexity.The taxonomic assignment revealed 7 archaeal (3.91%) and 172 bacterial (96.09%)MAGs.At the Phylum level, the microbiome spans 15 phyla with a striking majority of species assigned to Firmicutes (72.07%).The selecting pressure of stepwise increased ammonia levels shaped the microbial composition inducing a shift towards more resistant and specialised species.Among the methanogens, Methanoculleus bourgensis vb3066 appeared to play a dominant role (mean relative abundance (RA) = 4.33%), whereas the other six archaeal species have overall an average RA of 1.88% (Figure 2).SNVs in M. bourgensis vb3066 were mostly classified as synonymous (61.22%), while the remaining were nonsynonymous (27.98%), intergenic (10.45%), and multigenic (0.35%).The nsSNVs-impacted genes involved in hydrogenotrophic methanogenesis and mechanisms counteracting ammonia inhibition had a total length of 36246 (in F2) and 64113 (in P2) bp, and represented 2.01 and 3.55% of the genome in reactors F2 and P2, respectively (SI).These values are consistent with the analysis performed on the AD database 27 , where they don't exceed 10%.

Section S8: Observation of biofilm formation
Microbes inhabiting physiologically-unfavourable environments can form biofilms, establishing less stressing environmental niches, in which communities continuously thrive and evolve together 28 .The biofilm serves as a protective and homeostatic stronghold for resident microorganisms, allowing them to adapt to harsh environmental fluctuations 28 .Starting from the 19 th generation, biofilm-like structures in the form of pellicles or slimes were evidenced in the batch cultures for the first time (Figure S8).This formation was observed in all the following generations.It can be speculated that the biofilm presence favoured the survival of microorganisms in such stressing conditions.

Figure S2 :
Figure S2: Cumulative methane production for the reactors fed with casein (A) and those fed with glucose and acetate (B).Numbers after F and P identify the substrate: glucose (1), casein (2), acetate (3).

Figure S3 :Figure S4 :
Figure S3: Response of the most abundant MAGs to increasing ammonium concentration.From left to right the heatmaps show RA values (orange), the number of SNVs (blue) and the completeness of relevant KEGG modules (grey).Only the MAGs with RA equal to or higher than 3% in at least one generation are reported.

Figure S5 :
Figure S5: Frequency of nonsynonymous SNVs over time and strain deconvolution results for Acetomicrobium sp.mA.133.(A, D) filtered SNVs for reactors F2 and P2, respectively, (B, E) SNVs impacting proteins involved in the ammonia adaptation process, (C, F) abundance of strains calculated by STRONG, weighted on the results of the RA.Bold lines represent SNVs averages.

Figure S7 :*
Figure S7: PCA performed by integrating the biochemical parameters measured across generations (pH, TAN, FAN, TKN, VFA) with the RPKM values calculated from the reconstructed MAGs using CoverM v0.6.1.The distance used to plot the PCA was Bray-Curtis.

Table S2 :
Concentrations of NH 4 Cl and corresponding TAN used for the acclimation process.

Table S3 :
Genes with detected nsSNVs involved in the proposed mechanisms for ammonia resistance in M. bourgensis vb3066 and Acetomicrobium sp.ma133 and involved in methanogenesis in M. bourgensis vb3066.